Similarities of specificity and cofactor dependence in serum antiphospholipid antibodies from patients with human parvovirus B19 infection and from those with systemic lupus erythematosus

Objective. To assess the phospholipid specificity and immunoglobulin isotype of antiphospholipid (aPL) antibodies in patients with acute parvovirus B19 infection. Methods. Specificity of aPL and isotype distribution in the negatively charged phospholipids, cardiolipin and phosphatidyl serine, and in the neutral phospholipid, phosphatidyl ethanolamine, were measured in the sera of patients with acute parvovirus B19 infections (n = 12), in those with other acute viral infections (n = 10), and in those with syphilis (n = 15) by enzyme-linked immunosorbent assays. The dependence of anticardiolipin (aCL) binding on the presence of β₂-glycoprotein I (β₂-GPI) as a binding cofactor was assessed in these same groups, and was compared with sera from systemic lupus erythematosus (SLE) patients (n = 11) with raised aCL antibody reactivity. Results. Antibodies against any of the 3 phospholipids were found in all 3 groups of patients with infections (B19 in 11 of 12 patients, other viral infections in 8 of 10 patients, and syphilis in 14 of 15 patients). B19 patients' sera contained predominantly IgG antibodies against the negatively charged phospholipids, cardiolipin and phosphatidyl serine, and differed in their specificity and isotype distribution from those found in the other 2 patient groups. B19-associated aCL increased their binding to antigen in the presence of β₂-GPI as a binding cofactor, similar to aCL found in SLE patients, but unlike antibodies from patients with other viral infections or from those with syphilis. Conclusion. The results show the remarkable similarity in specificity of aPL antibodies between B19-infected patients and SLE patients, and raise the question of whether parvovirus infection may be a trigger for the development of aPL antibodies in autoimmune diseases.     

Some anticardiolipin antibodies recognize a combination of phospholipids with thrombin-modified antithrombin, complement C4b-binding protein, and lipopolysaccharide binding protein.

The standard enzyme-linked immunosorbent assay (ELISA) for anticardiolipin antibodies (ACA) detects a heterogenous group of antibodies against cardiolipin on its own, beta2-glycoprotein I (beta2GPI), and, potentially, other phospholipid-binding plasma proteins from bovine or human origin. In an attempt to identify new proteic targets of ACA, we selected 6 patients who possessed cofactor-dependent ACA but no antibody to human or bovine beta2GPI detectable in the beta2GPI-ELISA. Three of these samples proved to recognize beta2GPI in combination with cardiolipin, but not beta2GPI directly immobilized on gamma-irradiated polystyrene or agarose beads. In the other cases, the component required for ACA binding was purified from adult bovine serum or plasma by means of ammonium sulfate precipitation and chromatography on Phenyl-Sepharose, diethyl aminoethyl (DEAE)-cellulose, heparin-Ultrogel, and Sephacryl S-300 columns. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis coupled to N-terminal amino acid microsequencing identified the cofactors of patients no. 4, 5, and 6 ACA as lipopolysaccharide binding protein (LBP), complement C4b-binding protein (C4BP), and the thrombin-antithrombin (AT) complex, respectively. Adsorption of each of these cofactor preparations with cardiolipin liposomes led to suppression of ACA reactivity, concomitant with the loss of bands from SDS gels corresponding to sequenced material. Bacterial lipopolysaccharide (which forms high-affinity complexes with LBP) specifically neutralized the cofactor activity of the LBP preparation in a concentration-dependent manner. Bovine serum and plasma, as well as the C4BP preparation, optimally supported the binding of a rabbit anti-C4BP antiserum to immobilized cardiolipin. The binding of a rabbit anti-AT antiserum to solid-phase cardiolipin was sustained by the thrombin-AT preparation and bovine serum, but neither by bovine plasma nor by native AT, thus reproducing the behavior of patient no. 6 ACA. Taking advantage of the restricted recognition by the latter ACA of a cofactor from bovine origin appearing upon clotting, we studied the generation of such activity in human plasma supplemented with bovine AT or bovine prothrombin before clotting. In these conditions, patient no. 6 antibody binding to cardiolipin required the addition of bovine AT, whereas addition of bovine prothrombin alone was ineffective. We therefore concluded that those ACA targeted bovine AT once it has been modified/cleaved by thrombin. These findings underline the wide heterogeneity of ACA and the links that may exist between various coagulation pathways, inflammation and the complement system.     

Some ''antiphospholipid antibodies'' bind to β2-glycoprotein I in the absence of phospholipid

Some antiphospholipid antibodies (aPL) only bind to anionic phospholipids in the presence of a serum cofactor, beta 2-glycoprotein I (beta 2GPI). Whether these aPL can bind to beta 2GPI in the absence of phospholipid is controversial. We have purified anticardiolipin antibodies (aCL) from the plasma of four patients and beta 2GPI from normal plasma by solid phase affinity methods. All four aCL bound to cardiolipin and phosphatidylserine in the presence of beta 2GPI but not in its absence. The binding of two of the antibodies to cardiolipin and phosphatidylserine at various concentrations of human beta 2GPI was compared with that obtained using 10% bovine serum. The two antibodies responded differently to increasing beta 2GPI concentrations, and binding to phosphatidylserine was relatively greater than to cardiolipin using human beta 2GPI. All four aCL bound to plastic plates coated with beta 2GPI in the absence of phospholipid, and beta 2GPI in the fluid phase had no effect on binding. Binding to beta 2GPI coated plates was increased equally when bovine serum or bovine albumin were used as the sample diluent in place of gelatine. These findings and those of others have important implications for the design of assays for antiphospholipid antibodies.     

Anti-phospholipid antibodies are directed against a complex antigen that includes a lipid-binding inhibitor of coagulation: beta 2-glycoprotein I (apolipoprotein H).

Anti-phospholipid (aPL) antibodies that exhibit binding in cardiolipin (CL) ELISA can be purified to greater than 95% purity by sequential phospholipid affinity and ion-exchange chromatography. However, these highly purified aPL antibodies do not bind to the CL antigen when assayed by a modified CL ELISA in which the blocking agent does not contain bovine serum, nor do they bind to phospholipid affinity columns. Binding to the phospholipid antigen will only occur if normal human plasma, human serum, or bovine serum is present, suggesting that the binding of aPL antibodies to CL requires the presence of a plasma/serum cofactor. Using sequential phospholipid affinity, gel-filtration, and ion-exchange chromatography, we have purified this cofactor to homogeneity and shown that the binding of aPL antibodies to CL requires the presence of this cofactor in a dose-dependent manner. N-terminal region sequence analysis of the molecule has identified the cofactor as beta 2-glycoprotein I (beta 2GPI) (apolipoprotein H), a plasma protein known to bind to anionic phospholipids. These findings indicate that the presence of beta 2GPI is an absolute requirement for antibody-phospholipid interaction, suggesting that bound beta 2GPI forms the antigen to which aPL antibodies are directed. Recent evidence indicates that beta 2GPI exerts multiple inhibitory effects on the coagulation pathway and platelet aggregation. Interference with the function of beta 2GPI by aPL antibodies could explain the thrombotic diathesis seen in association with these antibodies.     

Antiphospholipid antibodies differ in aPL cofactor requirement.

Although autoimmune antiphospholipid antibodies (aPL) may require a serum cofactor, beta 2-glycoprotein I (beta 2GPI), for maximal binding in aPL ELISA, it is not known whether cofactor is absolutely required or is merely an enhancing factor for binding, nor is it clear whether aPL bind to cofactor itself, a cofactor-lipid complex, or a phospholipid modified in some way by cofactor. We therefore isolated and purified beta 2GPI and evaluated its relationship to both IgG and IgM aPL binding. aPL derived from different sera appear to have differing requirements for cofactor; the proportion of total binding attributable to cofactor varies from 46% to 95%. aPL do not bind to beta 2GPI in the absence of phospholipid. Enhanced binding to phospholipid is seen if beta 2GPI is provided either before or with the test antibody. Autoimmune aPL bind phospholipid better with human rather than bovine cofactor. The requirement for cofactor is greater for low-avidity aPL as measured in an IgG-human cofactor system. Cofactor requirement alone does not predict the presence or absence of associated clinical complications.     

β2-Glycoprotein i reactivity of monoclonal anticardiolipin antibodies from patients with the antiphospholipid syndrome

Objective. To elucidate the specificity of anticardiolipin antibodies (aCL) from patients with the antiphospholipid syndrome (APS) to various phospholipids (PLs), DNA, and β₂-glycoprotein I (β₂-GPI). Methods. Five monoclonal aCL were established from peripheral blood lymphocytes of 3 patients with the APS. The reactivity of monoclonal aCL with various PLs, with DNA, and with β₂-GPI was examined by enzyme-linked immunosorbent assay (ELISA). Results. All of the monoclonal aCL bound to anionic PLs, only in the presence of β₂-GPI. Neither monoclonal aCL nor β₂-GPI bound to DNA. Monoclonal aCL bound to solid-phase β₂-GPI on polystyrene ELISA plates that had carboxyl groups on their surface, but did not react with solid-phase β₂-GPI on ordinary polystyrene plates. A mixture of β₂-GPI and CL inhibited the binding of monoclonal aCL to β₂-GPI, but CL or β₂-GPI alone did not. Conclusion. Monoclonal aCL may recognize a cryptic epitope, which appears as a result of β₂-GPI binding to anionic PLs or to polystyrene with carboxyl groups.     

Antiphospholipid antibodies induced in mice by immunization with a cytomegalovirus‐derived peptide cause thrombosis and activation of endothelial cells in vivo

Objective

To characterize the binding and functional properties of antiphospholipid antibodies (aPL) induced by immunization with a viral peptide and to determine whether aPL are pathogenic in vivo.

Methods

Ten murine monoclonal aPL were generated from spleen cells of PL/J mice immunized with TIFI, a phospholipid‐binding peptide spanning Thr¹⁰¹–Thr¹²⁰ of ULB0‐HCMVA from human cytomegalovirus (CMV), which shares structural similarity with the phospholipid‐binding site of β₂‐glycoprotein I (β₂GPI).

Results

The antibodies generated had aPL activity that was inhibited by cardiolipin liposomes, and this inhibition was enhanced in the presence of β₂GPI. Some of the antibodies exhibited binding to cultured endothelial cells in vitro, and some had lupus anticoagulant activity. Injection with 2 of the monoclonal aPL in mice resulted in a significant increase in the number of leukocytes adhering to endothelial cells and enhanced thrombus formation in vivo.

Conclusion

These results indicate that aPL induced by immunization with a phospholipid‐binding CMV peptide are pathogenic in vivo. The results also suggest a mechanism (molecular mimicry) by which pathogenic aPL may be generated in patients with antiphospholipid syndrome.

     

Inhibition of prothrombin activation by antiphospholipid antibodies and β2-glycoprotein 1

Summary. Lupus anticoagulants, commonly found in the immunoglobulin fraction of patients with the antiphospholipid syndrome (APS), and the normal plasma protein β₂-glycoprotein 1 (β₂GP1) may both contribute to the in vitro impairment of prothrombin activation associated with the APS. We examined the effects upon prothrombin activation supported by phospholipid vesicles of plasma IgG preparations from APS patients in the presence and absence of β₂GP1. Using a purified system for measurement of prothrombin activation to thrombin, we demonstrated significant phospholipid concentration-dependent inhibition of prothrombin activation in the absence of β₂GP1 by 11 consecutive patient IgG preparations. The degree of inhibition of prothrombin activation by equivalent concentrations of patient IgG correlated well with the extent of prolongation of the plasma clotting time in lupus anticoagulant assays of whole patient plasma. Additional studies with eight patient IgG preparations indicated that the addition of β₂GP1 to patient IgG-phospholipid vesicle mixtures resulted in either independently additive inhibition by the two protein species (six cases) or potential inhibition of β₂GP1 of the IgG inhibitory activity demonstrable in the absence of β₂GP1 (two cases). In addition, β₂GP1-independent inhibition of prothrombin activation also occurred with three patient IgG preparations obtained by affinity binding to cardiolipin.     

Characterization of β2-glycoprotein I-dependent and -independent “antiphospholipid” antibodies from lupus-prone NZW/BXSB F1 hybrid male mice

Male (NZW × BXSB)F1 (W/BF1) mice develop a systemic lupus-like syndrome characterized by thrombocytopenia, coronary vascular disease, nephritis, and anticardiolipin antibodies. Three stable hybridoma cell lines secreting monoclonal anticardiolipin antibodies were developed from these mice by fusing their splenic lymphocytes with nonsecreting myeloma cell line, NS-1. Monoclonal antibody A1.17 reacted with cardiolipin in a β₂-Glycoprotein I-dependent manner. The epitope for this antibody consisted of β₂-glycoprotein I bound to cardiolipin or immobilized on plastic plates. Other anionic phospholipid-binding proteins, such as prothrombin or annexin V, had no significant effect in the reactivity of these antibodies. The specificity is similar to the autoimmune anticardiolipin antibodies described in patients with systemic lupus erythematosus and other infectious diseases. In contrast, monoclonal antibodies A1.72 and A1.84 reacted with cardiolipin in the absence of β₂-glycoprotein I. β₂-glycoprotein I, either in the fluid phase or bound to cardiolipin, inhibited the binding of these antibodies. The specificity of the latter two antibodies was similar to that described in patients with syphilis and allied disorders. Both types of antibodies had lupus anticoagulant properties. Thus lupus-prone male (NZW × BXSB)F1 (W/BF1) mice develop both β₂-glycoprotein I-dependent and β₂-glycoprotein I-independent anticardiolipin antibodies. Am. J. Hematol. 56:86–92, 1997. © 1997 Wiley-Liss, Inc.     

Rapid detection of anticardiolipin antibodies

A rapid screening method for the detection of antiphospholipid antibodies is described. Dense, red dyed polystyrene beads coated with cardiolipin were incubated with test sera for a short period of time, then added to a microtube containing anti-human IgG in a gel provided within a pre-cast card (DiaMed ID Microtyping System). The card was centrifuged at 150g for 5 min and then examined for movement of the beads through the gel. Beads without bound antibody travelled through the gel and formed a pellet on the bottom of the tube. Anti-human IgG within the gel matrix impeded cardiolipin-coated beads when antiphospholipid antibodies bound to the beads. Positivity was indicated by the formation of a layer of beads on the top of the gel matrix. Prospective analysis of 103 samples for the presence of antiphospholipid antibodies by flow cytometry and the gel-card technique showed good correlation between the two methods. All samples found to be positive by flow cytometry (23 of 103) were identified as positive by the gel-card technique. Two samples were identified as positive by the gel-card method but negative by flow cytometry. The technique is simple to perform and should prove useful as a rapid screening method for the detection of antiphospholipid antibodies. Am. J. Hematol. 57:315–319, 1998. © 1998 Wiley-Liss, Inc.     

Felty syndrome: autoimmune neutropenia or immune-complex-mediated disease?

Summary Immunofluorescence on polymorphonuclear cells (PMN) of patients with Felty syndrome (FS) revealed increased amounts of IgG, IgA, and IgM bound to the PMN surface compared with PMN of patients with rheumatoid arthritis alone. A positve correlation was found between the score for surface-bound immunoglobulins on FS-PMN and the results of the C1q binding assay in FS sera. After preincubation with sera from 20 patients with FS, immunofluorescence on PMN from healthy controls (HC) showed that these cells had bound IgG, IgA, and IgM. However F(ab')₂ fragments of IgG from FS sera did not bind to PMN, although the antigen-binding reactivity of the F(ab')₂ fragments was maintained as shown by control experiments. Immunoglobulins eluted from FS-PMN failed to bind to HC-PMN, whereas the corresponding IgG of patients with autoimmune neutropenia was bound. Gel filtration of FS sera on Sepharose 4B showed that the binding of IgG in FS sera to PMN did not coincide with the 7S peak but occurred mainly in fractions containing larger material. No binding of IgA and IgM to HC-PMN was found after incubation with FS sera pretreated with polyethylene glycol (PEG) to precipitate immune complexes. These results indicate that in sera of patients with FS the PMN-binding reactivity of IgG, IgA, and IgM is due to the binding of immune complexes containing these immunoglobulins and not to presence of autoantibodies directed to antigens on the neutrophil surface.     

The epitopes for some antiphospholipid antibodies are adducts of oxidized phospholipid and β2 glycoprotein 1 (and other proteins)

Circulating autoantibodies to phospholipids (aPLs), such as cardiolipin (CL), are found in patients with antiphospholipid antibody syndrome (APS). We recently demonstrated that many aPLs bound to CL only after it had been oxidized (OxCL), but not to a reduced CL analogue that could not undergo oxidation. We now show that the neoepitopes recognized by some aPLs consist of adducts formed between breakdown products of oxidized phospholipid and associated proteins, such as β₂ glycoprotein 1 (β₂GP1). Addition of human β₂GP1, polylysine, native low-density lipoprotein, or apolipoprotein AI to OxCL-coated wells increased the anticardiolipin antibody (aCL) binding from APS sera that first had been diluted so that no aCL binding to OxCL could be detected. No increase in aCL binding was observed when these proteins were added to wells coated with reduced CL. The ability of β₂GP1, polylysine, or low-density lipoprotein to be a “cofactor” for aCL binding to OxCL was greatly reduced when the proteins were methylated. Incubation of β₂GP1 with oxidized 1-palmitoyl-2-linoleyl-[1-¹⁴C]-phosphatidylcholine (PC), but not with dipalmitoyl-[1-¹⁴C]-PC, led to formation of covalent adducts with β₂GP1 recognized by APS sera. These data suggest that the reactive groups of OxCL, such as aldehydes generated during the decomposition of oxidized polyunsaturated fatty acids, form covalent adducts with β₂GP1 (and other proteins) and that these are epitopes for aCLs. Knowledge that the epitopes recognized by many aPLs are adducts of oxidized phospholipid and associated proteins, including β₂GP1, may give new insights into the pathogenic events underlying the clinical manifestations of APS.     

Anticardiolipin antibodies in chronic viral hepatitis. Do they have clinical consequences?

Antiphospholipid antibodies are a heterogeneous group of acquired autoantibodies that react with anionic phospholipids, such as cardiolipin. Anticardiolipin antibodies (ACAs) are frequently found in patients with systemic lupus erythematosus and other autoimmune disorders and can react with the phospholipid alone or bound to the cofactor beta2-glycoprotein-I. This latter form of cofactor-dependent ACAs is strongly associated with the occurrence of thrombotic events. ACAs have been observed to occur in both chronic hepatitis B and chronic hepatitis C as well as in other viral infections and in neoplastic diseases. In viral infection, ACAs are generally cofactor independent and may represent an epiphenomenon of the infection. Some studies, however, have found an increased incidence of thrombotic disorders in patients with chronic hepatitis C virus (HCV) who manifest ACA positivity suggesting that the presence of these autoantibodies may predispose to thrombosis in specific HCV-infected patients. In conclusion, ACAs are commonly found in patients with chronic viral infection but their pathogenetic role and the mechanisms that stimulate their production have not yet been clarified.     

Antiphospholipid antibody-dependent C5b-9 formation

The relationship between the presence of antiphospholipid antibodies (APA) and the production of the terminal membrane attack complex (MAC) of complement (C5b-9) was studied. Serum samples from known high positive APA patients induced platelet activation and destruction which was inhibited by heat-inactivation of the sera. The response was restored if the heat-inactivated APA-positive sera were supplemented with normal sera. Adsorption of the APA-positive sera with phospholipid (PL)-coated polystyrene beads inhibited platelet destruction. Addition of monoclonal antibody (mAb) to C5b-9 (aE11) also inhibited platelet destruction, suggesting that the APA-dependent platelet destruction might be complement-mediated. Purified APA, in the presence of normal serum, induced C5b-9 formation and binding to PL-coated beads in a dose-dependent manner as detected by flow cytometry. Prospective analysis of 200 serum samples for C5b-9 production showed that all sera testing negative for the presence of APA also tested negative for C5b-9 production. All sera with high levels of IgG binding to PL (GPL) showed evidence of C5b-9 production. Sera with low or moderate GPL values showed varying levels of C5b-9 production. These data suggest that complement may play a key role in APA-dependent platelet activation, in vivo .     

Significance of valine/leucine247 polymorphism of β2‐glycoprotein I in antiphospholipid syndrome: Increased reactivity of anti–β2‐glycoprotein I autoantibodies to the valine247 β2‐glycoprotein I variant

Objective

To clarify the consequences of the valine/leucine polymorphism at position 247 of the β₂‐glycoprotein I (β₂GPI) gene in patients with antiphospholipid syndrome (APS), by investigating the correlation between genotypes and the presence of anti‐β₂GPI antibody. The reactivity of anti‐β₂GPI antibodies was characterized using recombinant Val²⁴⁷ and Leu²⁴⁷ β₂GPI.

Methods

Sixty‐five Japanese patients with APS and/or systemic lupus erythematosus who were positive for antiphospholipid antibodies and 61 controls were analyzed for the presence of the Val/Leu²⁴⁷ polymorphism of β₂GPI. Polymorphism assignment was determined by polymerase chain reaction followed by restriction enzyme digestion. Recombinant Val²⁴⁷ and Leu²⁴⁷ β₂GPI were established to compare the reactivity of anti‐β₂GPI antibodies to β₂GPI between these variants. The variants were prepared on polyoxygenated plates or cardiolipin‐coated plates, and the reactivity of a series of anti‐β₂GPI antibodies (immunized anti‐human β₂GPI monoclonal antibodies [Cof‐19–21] and autoimmune anti‐β₂GPI monoclonal antibodies [EY1C8, EY2C9, and TM1G2]) and IgGs purified from patient sera was investigated.

Results

A positive correlation between the Val²⁴⁷ allele and the presence of anti‐β₂GPI antibodies was observed in the patient group. Human monoclonal/polyclonal anti‐β₂GPI autoantibodies showed higher binding to recombinant Val²⁴⁷ β₂GPI than to Leu²⁴⁷ β₂GPI, although no difference in the reactivity of the immunized anti‐β₂GPI between these variants was observed. Conformational optimization showed that the replacement of Leu²⁴⁷ by Val²⁴⁷ led to a significant alteration in the tertiary structure of domain V and/or the domain IV–V interaction.

Conclusion

The Val²⁴⁷ β₂GPI allele was associated with both a high frequency of anti‐β₂GPI antibodies and stronger reactivity with anti‐β₂GPI antibodies compared with the Leu²⁴⁷ β₂GPI allele, suggesting that the Val²⁴⁷ β₂GPI allele may be one of the genetic risk factors for development of APS.

     

Endothelial cells as target for antiphospholipid antibodies. Human Polyclonal and Monoclonal Anti-β2-Glycoprotein I Antibodies React In Vitro with Endothelial Cells Through Adherent β2-Glycoprotein I and Induce Endothelial Activation

Objective. To investigate the ability of human anti-β₂-glycoprotein I (anti-β₂GPI) antibodies to recognize the cofactor adherent on endothelial cells (EC) and to modulate endothelial functions. Methods. Six human affinity-purified polyclonal anti-β₂GPI IgG and 2 IgM monoclonal antibodies (MAb) were obtained from patients with the antiphos-pholipid syndrome. The antibodies were tested for their ability to 1) bind to endothelial monolayers through the adherent β₂GPI and 2) modulate endothelial adhesion molecule expression and interleukin-6 (IL-6) and 6-keto-prostaglandin F_1α (6-keto-PGF_1α) secretion. Results. The affinity-purified IgG and the MAb with anti-β₂GPI activity, but not the respective controls, displayed EC binding, which declined on cells incubated in serum-free medium and was restored in a dose-dependent manner by exogenous human β₂GPI. After EC binding, both polyclonal and monoclonal antibodies up-regulated adhesion molecule expression. Anti-β₂GPI MAb also significantly increased IL-6 and 6-keto-PGF_1α secretion. Conclusion. These findings support the hypothesis that anti-β₂GPI antibodies bind and activate EC through the adherent cofactor β₂GPI, likely leading to a procoagulant state.     

The BH1 idiotype defines a population of anticardiolipin antibodies closely associated with the antiphospholipid syndrome.

BACKGROUND: A human IgM monoclonal anticardiolipin antibody - BH1 - has previously been described, which has characteristics typical of antiphospholipid antibodies in the serum of patients with antiphospholipid syndrome (APS). It appears to be idiotypically distinct from other human monoclonal autoantibodies of different or overlapping ligand-binding specificities derived from patients with related conditions. AIM: To determine whether the idiotype of BH1 is expressed on particular populations of antibodies (antiphospholipid and anti-beta2-glucoprotein I) in the serum of patients with APS and other conditions. METHODS: Sera from patients with APS (9), systemic lupus erythematosus without APS ('uncomplicated SLE' -9), and rheumatoid arthritis (RA 15), and from normal controls (15) were tested by enzyme-linked immunosorbent assay (ELISA) for reactivity with cardiolipin, beta2 glycoprotein I (beta2GPI), and a polyclonal anti-idiotype raised against BH1 (RIdBH1). Absorption experiments were subsequently performed on selected sera using micelles of cardiolipin or phosphatidyl choline. RESULTS: Eight out of nine patients with APS were positive for binding to RIdBH1 (IgG and/or IgM), while only one patient with uncomplicated SLE and none of the patients with RA or the healthy controls were positive. Although all of the patients with APS were positive for binding to beta2GPI, there was poor correlation between these results and levels of binding to cardiolipin and RIdBH1. Absorption of sera from patients with APS by cardolipin micelles resulted in a median reduction in IgG anticardiolipin and anti-beta2GPI activity of 81.6% and 6.3% respectively. For those sera positive for IgG reactivity with RIdBH1 the median reduction in this activity was 79.4%. Antibodies eluted from selected micelles showed activity against cardiolipin, beta2GPI and RIdBH1. Three anticardiolipin-positive sera from patients with RA were similarly absorbed; however the eluted antibodies failed to bind to RIdBH1. Absorption of all these sera with phosphatidyl choline resulted in no significant reduction in any of these activities. CONCLUSIONS: The BH1 idiotype defines a population of serum antibodies associated with features of APS. The antibody response in this condition, though diverse, may include the expression of a restricted group of variable region genes.     

Plasmin immunization preferentially induces potentially prothrombotic IgG anticardiolipin antibodies in MRL/MpJ mice

Objective

To test the hypothesis, utilizing 2 experimental mouse models, that plasmin is an important autoantigen that drives the production of certain IgG anticardiolipin (aCL) antibodies in patients with the antiphospholipid syndrome.

Methods

BALB/cJ and MRL/MpJ mice were immunized with Freund's complete adjuvant in the presence or absence of human plasmin. The mouse sera were analyzed for production of IgG antiplasmin, IgG aCL, and IgG anti–β₂‐glycoprotein I (anti‐β₂GPI) antibodies. IgG monoclonal antibodies (mAb) were generated from the plasmin‐immunized MRL/MpJ mice with high titers of aCL, and these 10 mAb were studied for their binding properties and functional activity in vitro.

Results

Plasmin‐immunized BALB/cJ mice produced high titers of IgG antiplasmin only, while plasmin‐immunized MRL/MpJ mice produced high titers of IgG antiplasmin, IgG aCL, and IgG anti‐β₂GPI. Both strains of mice immunized with the adjuvant alone did not develop IgG antiplasmin or IgG aCL. All 10 of the IgG mAb bound to human plasmin and cardiolipin, while 4 of 10 bound to β₂GPI, 3 of 10 bound to thrombin, and 4 of 10 bound to the activated coagulation factor X (FXa). Functionally, 4 of the 10 IgG mAb inhibited plasmin activity, 1 of 10 hindered inactivation of thrombin by antithrombin III, and 2 of 10 inhibited inactivation of FXa by antithrombin III.

Conclusion

Plasmin immunization leads to production of IgG antiplasmin, aCL, and anti‐β₂GPI in MRL/MpJ mice, but leads to production of only IgG antiplasmin in BALB/cJ mice. IgG mAb generated from plasmin‐immunized MRL/MpJ mice bind to various antigens and exhibit procoagulant activity in vitro. These results suggest that plasmin may drive potentially prothrombotic aCL in genetically susceptible individuals.

     

Equilibrium and Kinetics of Sin Nombre Hantavirus Binding at DAF/CD55 Functionalized Bead Surfaces

Decay accelerating factor (DAF/CD55) is targeted by many pathogens for cell entry. It has been implicated as a co-receptor for hantaviruses. To examine the binding of hantaviruses to DAF, we describe the use of Protein G beads for binding human IgG Fc domain-functionalized DAF ((DAF)₂-Fc). When mixed with Protein G beads the resulting DAF beads can be used as a generalizable platform for measuring kinetic and equilibrium binding constants of DAF binding targets. The hantavirus interaction has high affinity (24–30 nM; k_on ~ 10⁵ M⁻¹s⁻¹, k_off ~ 0.0045 s⁻¹). The bivalent (DAF)₂-Fc/SNV data agree with hantavirus binding to DAF expressed on Tanoue B cells (K_d = 14.0 nM). Monovalent affinity interaction between SNV and recombinant DAF of 58.0 nM is determined from competition binding. This study serves a dual purpose of presenting a convenient and quantitative approach of measuring binding affinities between DAF and the many cognate viral and bacterial ligands and providing new data on the binding constant of DAF and Sin Nombre hantavirus. Knowledge of the equilibrium binding constant allows for the determination of the relative fractions of bound and free virus particles in cell entry assays. This is important for drug discovery assays for cell entry inhibitors.     

Heterogeneity of anticardiolipin antibody

The antibody to cardiolipin(ACA) was tested in patients with systemic rheumatic disease. The frequency of IgG ACA was 46/100(46.0%) in systemic lupus erythematosus(SLE). In other rheumatic disease, this was less than 20%. Significant correlation between the presence of IgG ACA and thrombosis and/or thrombocytopenia was found in patients with SLE. Eight sera containing high titered IgG ACA from lupus patients were selected for further inhibition study. Inhibitors were consisted of cardiolipin(CL), phosphatidyl(p-)serine, p-inositol, p-glycerol, p-ethanolamine, p-choline, ds-DNA, ss-DNA, fresh platelets(PLT)and fresh red blood cells(RBC). All sera were markedly inhibited by negatively charged phospholipids. In 4 sera(group B), there was moderate inhibition by ss-DNA, ds-DNA, PLT and RBC. In another 4 sera(group A), mild but significant inhibition was obtained by PLT alone. The number of platelet in group A was less than that in group B. There were some differences in inhibitory activity, suggesting heterogeneity of antibody to CL. It may be possible to speculate that heterogeneity of IgG ACA cause various combination of clinical features such as thrombocytopenia and thrombosis.